Ovulation is one of the most fundamental events in reproduction and requires a dramatic surge of luteinizing hormone (LH) released by the anterior pituitary gland into the peripheral circulation. The secretion of LH from the pituitary is cued by the hypothalamic neuropeptide, gonadotropin-releasing hormone (GnRH). Thus, the secretion of GnRH from hypothalamic neurons and its interpretation by gonadotropes in the pituitary constitute the essential central events for reproductive competence. Thus, understanding and, ultimately, controlling the mechanisms underlying the generation of the preovulatory LH surge remains a primary focus of research in reproductive endocrinology at both the basic and clinical levels. It has become almost axiomatic that estradiol- 172 (E2) secreted from the preovulatory follicle serves as the most proximate regulator of the LH surge via two sites of action. First, E2 enhances sensitivity of the pituitary by increasing GnRH receptor expression. Second and temporally delayed, it stimulates increased hypothalamic secretion of GnRH. As these 2 physiological sequelae to elevated E2 have been known for decades, it is troubling that our understanding of the underlying mechanisms at both the level of GnRH secretion (hypothalamic) and GnRH responsiveness (pituitary) remains quite undeveloped. In regard to the latter, the lack of progress reflects the inability of in vitro models (cell lines) and promoters (mouse GnRH receptor gene) to recapitulate the increase in pituitary responsiveness to GnRH induced by E2. In contrast, using transgenic mouse models we have demonstrated that the proximal promoter of the ovine GnRH receptor gene is highly E2 responsive. This E2 responsive phenotype does not; however, appear to involve direct binding of activated estrogen receptor (ER) to the sheep gene. Rather, we find that membrane impermeable conjugates of E2 regulate LH secretion in sheep and increase GnRH receptor expression. As such, heightened responsiveness of the pituitary preceding the preovulatory LH surge may reflect E2 signaling through a membrane site of action - this is the central biological issue that exists as the core of our proposed research. Accordingly, using both in vivo and in vitro tests of promoter function and parallel analyses of the endogenous ovine GnRH receptor gene we will determine the hierarchy of ER subtypes that underlie E2 responsiveness of GnRH receptor expression, the initial cellular site of action of E2 that elicits enhanced expression of GnRH receptors and the identity of the regulatory element(s) that mediate the transcriptional response of the GnRHR gene to E2. Understanding regulation of GnRH and its receptor is important for fertility control; however, GnRH agonists and antagonists are used to treat fibroid tumors, endometriosis, and reproductive tissue carcinomas thus underscoring the need for a full understanding of GnRH and the GnRH receptor in both health and disease. PUBLIC HEALTH RELEVANCE: These studies will refine our understanding of estrogen signaling in the anterior pituitary gland (the master endocrine gland) as well novel mechanisms underlying estrogen regulation of important physiological targets throughout the body. Understanding these mechanisms is critical to the development of new generations of both pro and anti-estrogen-like pharmaceuticals that are increasingly specific for the desired biological effect. Understanding physiological regulation of reproductive hormones like gonadotropin releasing hormone (GnRH) and the GnRH receptor is important to fertility regulation; however, the application of potent GnRH agonists and antagonists in the treatment of uterine fibroids, endometriosis and reproductive tissue carcinomas underscores the importance of this topic in both health and disease.